Biological metabolism in living beings is a reaction mechanism with high matrix selectivity and remarkably high efficiency. It is characterized in reaction progressing in a relatively moderate atmosphere held at the room temperature and neutral. Biological metabolism herein pertains involves aspiration and photosynthesis, among others, which converts oxygen and various kinds of nutritional elements such as sugars, fats, oils, and proteins to energy necessary for growth of microorganisms and cells.
Such biological reactions largely rely on biological catalysts made of proteins, namely, enzymes. The idea of using the catalytic reaction of enzymes has been put into practice from early days of the history of humankind. Its applications range over many industrial fields including brewing industry, fermentation industry, fiber industry, leather industry, food industry and drug industry. Recently, its applications to the field of electronics such as biosensors, bioreactors, bio-fuel cells, which incorporate the catalytic function into electrode systems, have come to be examined and brought into practice.
Enzymes are composed of proteins, and usually instable to heat, strong acids, strong alkalis, organic solvents, and the like. Therefore, enzymes could be used only in water solutions. To bring about enzymatic reaction, the batch process has been used heretofore, which dissolves an enzyme into a water solution to have it act on the matrix. However, there is an extreme technical difficulty in recovery and re-use of enzymes out of the solution after reaction without degeneration of the enzymes. Thus, the method results in disposing of enzymes after each reaction, and this must be an uneconomical way of use.
Under the background, there is a proposal of water-insoluble immobilization enzymes. Once being insolubilized to water, highly specific enzymes can be handled in the same manner as solid catalysts used in ordinary chemical reactions. This is a very useful way of use of enzymes.
Much the same is true on application of enzymes to electrode systems. High-density immobilization of an enzyme on an electrode makes it possible to efficiently catch enzymatic reactions occurring near the electrode as electric signals. For researches of electrode systems, in general, it is necessary to use an electron acceptor compound behaving as an electron transfer medium between the protein as the enzyme and the electrode, where intermediation of electrons is unlikely to occur. Preferably, this electron acceptor compound is immobilized similarly to the enzyme.
Methods of immobilizing enzymes are roughly classified to two groups, i.e. entrapping methods and the binding methods. Entrapping methods are further classified to lattice-type one and microcapsule type one. On the other hand, binding methods are further classified to the absorption method, covalent binding method (cross-linking method), and others, depending upon the binding mode.
The entrapping method envelops the enzyme with a water-insoluble, semi-permeable polymer not binding to the enzyme itself. A merit of this method is that the possibility of damaging the enzymatic activity is low because the immobilization takes place under a relatively mild condition. On the other hand, since the enzyme are not eluted upon immobilization while the reaction matrix of the enzyme must have voids easy to pass, it is necessary to select an appropriate entrapping agent every time when the combination of the enzyme and the matrix varies.
The absorption method uses ionic absorption or physical absorption of enzymes. Although this method employs easy ways of immobilization, the state of absorption is susceptible to service conditions, and absorption and desorption of enzymes tend to become instable. Therefore, this method cannot be a general technique for immobilization.
The covalent binding method uses amino groups, carboxyl groups of enzymes for binding by the use of a cross-linking agent. Although this method can stably immobilize enzymes relatively easily, it often invites inactivation of the enzyme because the cross-linking agent may modify a portion near the active center of the enzyme or the conditions for cross-linking may be severe for the enzyme.
A manufacturing method of a functional electrode has been proposed, which impregnates a porous electrode with a monomer for generating a conductive polymer and a supporting electrolyte, and brings about electrolytic oxidization in the support electrolyte solution to make a conductive polymer coat on the entire surface inside the porous electrode (Japanese Patent Laid-open Publication No. JP-H06-271655). In addition, a method of measuring optical isomers such as D-lactic acid, L-lactic acid, and so on, has been proposed (Japanese Patent Laid-open Publication No. JP-H06-121697).
As reviewed above, those conventional immobilization methods have respective demerits, and an effort is required to determine an immobilization method optimum for each enzyme-matrix combination. Furthermore, upon determining immobilization to electrode systems, since immobilization of the electron acceptor compound as the electron transport medium is also desirable as mentioned above, here is required immobilization capable of retaining respective abilities of the enzyme, electron acceptor compound and matrix, which makes determination of an immobilization method more difficult.
It is therefore an object of the invention to provide an immobilization carrier suitable for realizing a highly efficient functional electrode incorporating catalytic function of an enzyme by immobilizing the enzyme to an electrode system or immobilizing the enzyme and an electron acceptor compound simultaneously, an electrode function-utilizing device and their manufacturing methods.